Method for purifying protein and glucose dehydrogenase

ABSTRACT

A method of purifying a target protein from a solution, in which the target protein containing an electron transfer protein is dissolved, with the use of liquid chromatography. The liquid chromatography is performed by introducing the above-described protein solution into a tank filled with a packing agent, thus bonding the target protein to the packing agent, removing impurities, and then eluting the target protein from the packing agent with the use of an eluent containing a hydroxycholanoic acid salt. As an example of the above protein, glucose dehydrogenase containing a protein having an activity of dehydrogenating glucose is cited. The liquid chromatography is performed by combining hydrophobic chromatography with anion exchange chromatography.

TECHNICAL FIELD

The present invention relates to a method for purifying protein by usingliquid chromatography. The method is used, for example, when purifyingglucose dehydrogenase which is bonded to electron transfer protein.

BACKGROUND ART

Development efforts have been made in many different fields of industryfor biosensors incorporating an enzyme which makes a specific reactionwith a specific substrate. A representative example of such biosensorsis a glucose sensor utilized mainly in the field of medical care.

The glucose sensor establishes a reaction system which includes anenzyme and an electron transfer material. When using the glucose sensor,an amperometric method for example is employed to quantitate glucose.The enzyme is provided by e.g. glucose oxidase (GOD) and glucosedehydrogenase (GDH).

GOD has high substrate specificity to glucose, high thermal stability,and is less expensive than other enzymes because it can be mass-producedindustrially. A shortcoming on the other hand is that reaction systemsinvolving GOD are highly sensitive to oxygen dissolved in the sample,and so the dissolved oxygen can affect the measurement.

On the other hand, reaction systems involving GDH are not susceptible todissolved oxygen in the sample. For this reason, reaction systems whichutilize GDH allow accurate measurement of the glucose level even if themeasurement is made in an environment poor in oxygen partial pressure,or if the measurement is made to a high-concentration sample which has ahigh oxygen demand. Shortcomings of GDH include poor thermal stabilityand lower substrate specificity than GOD.

Under these circumstances, efforts have been made in search for anenzyme which covers the shortcomings of both GOD and GDH. As disclosedin the International Disclosure WO02/36779, Hayade separated a newstrain (Burkholderia cepacia KS1) from soil near a hot spring, andobtained a new GDH from the strain. This GDH included α, β, γ subunits(hereinafter called “CyGDH”), had a high rate of reaction with electrontransfer materials, and sufficient thermal stability, and so wassuitable for use in glucose sensors.

When utilizing CyGDH in glucose sensors, CyGDH must be purified from anenzyme solution which contains CyGDH. Enzyme is usually purified bymeans of liquid chromatography, so the inventor et al. of the presentinvention followed a common method of hydrophobic chromatography andanion exchange chromatography in an attempt to process the enzymesolution. However, it was not possible to purify CyGDH to a high levelof purification, as SDS-PAGE examinations revealed that the obtainedenzyme solution contained a number of different proteins in addition toα, β, γ subunits.

DISCLOSURE OF THE INVENTION

The present invention aims at providing a new method for purifying aprotein.

A first aspect of the present invention provides a method for purifyinga target protein from a protein solution which contains the targetprotein, by using liquid chromatography. The liquid chromatographyincludes: a first step of introducing the protein solution into a columnwhich is filled with a packing agent and causing the packing agent tohold the target protein; and a second step of eluting the target proteinby using an eluent which contains a hydroxy-cholate.

It should be noted here that in the present specification, that the term“liquid chromatography” refers, unless otherwise specified, to both of acolumn method in which the purification is made in a flow(continuously), and a batch method of purifying protein. An example ofthe batch method is placing a packing agent and the protein solutiontogether in a column to cause the packing agent to bind the targetprotein, then separating the packing agent, and then bringing thepacking agent in contact with the eluent thereby separating andcollecting the target agent from the packing agent.

An example of the target protein, which is the object of thepurification, is a protein which contains electron transfer protein. Atypical example of the target protein is glucose dehydrogenase whichcontains an electron transfer protein and a protein that has glucosedehydrogenation activity.

The packing agent can be an ion-exchange gel, and the protein ispurified by means of an ion-exchange chromatography. In this case, theion-exchange gel contains a quaternary ammonium group as an ion-exchangegroup.

Here, the term “hydroxy-cholate” refers to salts of cholic acid whichare trihydrates of cholanic acids, as well as their derivatives in abroad sense. Examples of hydroxy-cholates include cholates,glicoursodeoxycholate, tauroglicoursodeoxycholate,tauroursodeoxycholate, ursodeoxycholate, glycocholate, taurocholate,glycochenodeoxycholate, taurochenodeoxycholate, glycodeoxycholate,taurodeoxycholate, chenodeoxycholate, deoxycholate, glycolithocholate,taurolithocholate, and lithocholate. Among others, it is preferable touse a cholate such as sodium cholate.

When eluting the target protein from the packing agent, preferably, theconcentration of the hydroxy-cholate in the eluent is maintained at aconstant level. In this case, the concentration of the hydroxy-cholatein the eluent is preferably selected from a range of 0.5 through 2.5 wt%, and more preferably, from 0.8 through 1.2 wt %. Such a method isapplied not only to the elution of the target protein in a batch processbut also to the elution of the target protein in a continuous process.Alternatively, the concentration of the hydroxy-cholate in the eluentmay be varied with time, in eluting the target protein. In this case,the upper limit concentration of the eluting agent should be not higherthan 3 wt % for example, and more preferably not higher than 1.5 wt %,and most preferably not higher than 1 wt %. In other words, theconcentration of the eluent is varied within the range of 0-3 wt % forexample, more preferably within the range of 0-1.5 wt %, and mostpreferably within the range of 0-1.0 wt %.

The electron transfer protein has a molecule weight of e.g.approximately 43 kDa in SDS-gel electrophoresis under a reducingenvironment. On the other hand, the protein which has glucosedehydrogenation activity has a molecule weight of approximately 60 kDain SDS-gel electrophoresis under a reducing environment. Glucosedehydrogenase which contains such electron transfer protein and subunitsis obtainable from e.g. a microorganism belonging to the genusBurkholderia which is capable of producing the glucose dehydrogenase, orfrom a transformant thereof.

There is no specific limitation to the microorganism of the genusBurkholderia which is used in the present invention, as long as themicroorganism is capable of producing the target enzyme. Preferably,however, Burkholderia cepacia, and Burkholderia cepacia KS1 strain(hereinafter simply called “KS1 strain”) in particular, is preferred.KS1 strain was deposited as FERM BP-7306, on Sep. 25, 2000, with theInternational Patent Organism Depositary (IPOD), National Institute ofAdvanced Industrial Science and Technology (AIST) (Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566).

The transformant can be produced, for example, by engineering a hostmicroorganism with a DNA from a microorganism belonging to the genusBurkholderia for coding the electron transfer protein and the proteinactive against glucose. The host microbe is preferably a microorganismbelonging to the genus Pseudomonas (Pseudomonas putida in particular) orE. coli bacterium.

As disclosed in International Publication WO02/36779 and others, glucosedehydrogenase derived from KS1 strain contains a subunit (α subunit) andan electron transfer protein (β subunit), and γ subunit which has amolecular weight of approximately 14 kDa in the SDS gel-electrophoreticmigration under reducing environment.

Hayade has confirmed that higher enzyme activity is achieved by acombination of γ subunit and α subunit than by α subunit only.Therefore, in view of enzyme activity, it is preferable to manifest γsubunit, and when engineering the DNA, γ subunit structural gene shouldpreferably be included in an upstream region of α subunit. Then, whenthe transformant produces α subunit, γ subunit which has been manifestedalready and existing as a protein will promote efficient production of αsubunit in the microorganism.

A second aspect of the present invention provides a method for purifyingglucose dehydrogenase using a combination of hydrophobic chromatographyand anion exchange chromatography. The hydrophobic chromatographyincludes: a step of causing a stationary phase to hold the glucosedehydrogenase; a step of eluting unnecessary proteins; and a step ofeluting the glucose dehydrogenase by using an eluent containing ahydroxy-cholate. The anion exchange chromatography includes: a step ofcausing a stationary phase to hold the glucose dehydrogenase; and a stepof eluting the glucose dehydrogenase by using an eluent containing ahydroxy-cholate.

In the hydrophobic chromatography, elution of the glucose dehydrogenasefor example, is performed by varying the concentration of thehydroxy-cholate in the eluent with time. On the other hand, in the anionexchange chromatography, elution of the glucose dehydrogenase forexample, is performed by keeping the concentration of thehydroxy-cholate in the eluent. Alternatively, the concentration of thehydroxy-cholate in the eluent may be varied with time, in eluting thetarget protein. In this case, the upper limit concentration of theeluting agent should be not higher than 3 wt % for example, and morepreferably not higher than 1.5 wt %, and most preferably not higher than1 wt %. In other words, the concentration of the eluent is varied withinthe range of 0-3 wt % for example, more preferably within the range of0-1.5 wt %, and most preferably within the range of 0-1.0 wt %.

In the method for purifying glucose dehydrogenase according to thepresent invention, preferably, the anion exchange chromatography iscarried out after the hydrophobic chromatography.

The glucose dehydrogenase is produced by e.g. a microorganism belongingto the genus Burkholderia. An example of the microorganism belonging tothe genus Burkholderia is KS1 strain.

The glucose dehydrogenase may be made by a transformant. Thetransformant can be produced by engineering a host microorganism with aDNA from a microorganism belonging to the genus Burkholderia for codingthe glucose dehydrogenase. The host microbe can be Pseudomonas putida orE. coli bacterium for example.

In the method for glucose dehydrogenase according to the presentinvention, preferably, the anion exchange chromatography is performed byusing an ion-exchange gel which contains a quaternary ammonium group asan ion-exchange group, and the hydroxy-cholate is provided by a cholate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an SDS-PAGE result of an enzyme which was purified with asodium cholate eluent from a crude enzyme solution obtained from atransformant of Pseudomonas putida.

FIG. 2 shows an SDS-PAGE result of an enzyme which was purified with aNaCl or KCl eluent from a crude enzyme solution obtained from thetransformant of Pseudomonas putida.

FIG. 3 shows an SDS-PAGE result of an enzyme which was purified with asodium cholate eluent from a crude enzyme solution obtained from atransformant of E. coli bacterium, together with results of two enzymes;one of which was purified from a crude enzyme solution obtained from atransformant of Pseudomonas putida while the other was purified from acrude enzyme solution obtained from KS1 strain.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, methods of purifying a protein according to the presentinvention will be described specifically, taking an example of purifyingglucose dehydrogenase (herein after called “GDH”).

When purifying glucose dehydrogenase, first, an enzyme solution isprepared. The enzyme solution may be obtained from a microbe whichproduces the glucose dehydrogenase, a culture of the microbe, atransformant derived through insertion of DNA which is extracted fromthe microbe, or a culture of the transformant.

If GDH is inside the microbe, the enzyme solution can be obtained byfirst separating the microbe from the culture through filtration,centrifugation, etc., and then fragmentizing the microbe mechanically orenzymatically with e.g. lysozyme, followed as necessary, bysolubilization of GDH through addition of a chelating agent such as EDTAand a surfactant. On the other hand, if GDH is outside the microbe (inthe culture), the enzyme solution can be obtained by separating themicrobe from the culture through filtration, centrifugation and so on.

Among microbes which produce glucose dehydrogenase, microbes of thegenus Burkholderia, and Burkholderia cepacia in particular is preferablyutilized. From Burkholderia cepacia, it is possible to obtain the enzymesolution in the form of a solubilized membrane fraction for example. Inorder to obtain the solubilized membrane fraction, for example, firstthe microbe is centrifugally separated from a culture, and the microbeis fragmentized to obtain a cell extract. The cell extract is thencentrifuged, and thereafter, obtained supernatant fluid issuper-centrifuged. The resulting sediment is the solubilized membranefraction. The fragmentization of the microbe can be made in an ordinarymechanical or enzymatic method.

The transformant can be produced by first obtaining DNA which codesmanifestation of e.g. α subunit (a protein having glucosedehydrogenating activity) and β subunit (electron transfer protein), andthen introducing the DNA into a host microbe by using a recombinantvector.

When obtaining the DNA, first, a recombinant vector is constructed. Theconstruction of recombinant vector can be achieved by first separatingand purifying a chromosome DNA from a microbe which produces an enzymehaving glucose dehydrogenation activity, preparing the chromosome DNAfragments through shredding or PCR amplification, and then binding andclosing the chromosome DNA fragments with a linear expression vector.

Examples of the host microbe include E. coli and other enteric bacteria,gram-negative bacteria such as the genus pseudomonas and the genusGluconobacter, gram-positive bacteria including the genus Bacillus suchas Bacillus subtilis, yeasts such as Saccharomyces cerevisiae, andfilamentous bacteria such as Aspergillus niger. Among these, E. colibacteria and those of the genus pseudomonas (pseudomonas putida forexample) are preferably used. The transformation of the microbes can bemade by competent cell method through calcium treatment for the genusEscherichia. Protoplast methods can be used for the genus bacillus, KUmethod and KUR method for yeasts, and micromanipulation methods forfilamentous bacteria. Transformation can also be made by usingelectropolation method.

The enzyme solution is purified by using liquid chromatography. Inperforming the purification through liquid chromatography, a selectionis made for the type, number and combination of different types ofchromatography so that a target level of purification can be achieved.Usable types of liquid chromatography include gel filtrationchromatography, adsorption chromatography, ion exchange chromatographyand affinity chromatography.

The liquid chromatography may be made by first having the target proteincaptured in the stationary phase in the column, and then supplyingeluent continuously thereby eluting the target protein. Alternatively, abatch method may be used. In the batch method, for example, a column issupplied with a packing agent and a solution which contains a targetprotein so the packing agent will hold the target protein. Then,impurities are removed, and eluent is supplied to elute the targetprotein from the packing agent for collection.

The eluent is a solution which contains a hydroxy-cholate as an elutingagent. If the liquid chromatography is performed for a plurality oftimes before yielding the purified target protein, hydroxy-cholate isused as the eluent at least in one cycle of the liquid chromatography.In this case, preferably, hydroxy-cholate is used in the last cycle ofliquid chromatography, as the eluent.

Examples of hydroxy-cholate include cholates, glicoursodeoxycholate,tauroglicoursodeoxycholate, tauroursodeoxycholate, ursodeoxycholate,glycocholate, taurocholate, glycochenodeoxycholate,taurochenodeoxycholate, glycodeoxycholate, taurodeoxycholate,chenodeoxycholate, deoxycholate, glycolithocholate, taurolithocholateand lithocholate. Among others, use of cholate, such as sodium cholateis preferred.

When bringing the eluent into contact with the packing agent, theconcentration of the hydroxy-cholate in the solvent may be kept at aconstant level, or the hydroxy-cholate concentration may be linearlychanged with the time during the supply.

The liquid chromatography may be made directly to the enzyme solution,or may be made after the target protein in the enzyme solution has beenconcentrated. The concentration can be made by e.g. vacuumconcentration, membrane concentration, salting-out procedure, factionalprecipitation using hydrophilic solvent (e.g. methanol, ethanol andacetone), heating, and isoelectric process.

The purified enzyme thus obtained, is made into a powdery productthrough such a process as freeze-drying, vacuum-drying and spray-drying,for distribution in the market.

EXAMPLES

Hereinafter, specific examples of the fabrication methods outlined abovewill be described, with demonstration that GDH can be purified moreefficiently when the eluent is provided by sodium cholate than when theeluent is provided by NaCl or KCl.

<Obtainment of Enzyme Solution>

(1) Obtainment of Enzyme Solution from Burkholderia Cepacia KS1 Strain

Burkholderia cepacia KS1 strain was cultured under an aerobic condition.More specifically, the KS1 strain was cultured in 20 L of medium at 34°C. for 8 hours. The medium contained ingredients listed in Table 1, perliter. TABLE 1 MEDIUM COMPOSITION Polypeptone 10 g Yeast extract 1 gNaCl 5 g KH₂PO₄ 2 g Glucose 5 g Einol (ABLE Co., Tokyo Japan) 0.14 gTotal, distilled water 1 L pH 7.2

Next, the 20 L of culture medium was centrifuged at 4° C., for 10minutes at 9000×g, to obtain approximately 250 g of microbe body. Thecollected microbe body was frozen, then suspended in 10 mM phosphatebuffer solution (pH6), and processed in a high-pressure homogenizer(manufactured by Rannie, Denmark) several times at a pressure of 500bar, to fragmentize cell membrane. The cell extract obtained by cellmembrane fragmentation showed a GDH activity of 60 kU. The cell extractwas then centrifuged under 8000×g for 60 minutes, to remove cell solid.Further, the supernatant liquid was super-centrifuged at 10° C. under170,000×g for an hour, to collect membrane fraction as the sediment.

The membrane fraction was dissipated in 10 mM phosphate buffer solution(pH 6), so as to have a final sodium cholate concentration of 1.5%, andKCl concentration of 0.1 M, and steered at 4° C. over a night. As aresult, membrane fraction suspension which contained GDH at 30 kU wasobtained. The membrane fraction suspension was super-centrifuged at 10°C. under 170,000×g for 90 minutes, to remove sediment, and to obtainGDH-containing solubilized membrane fraction (at GDH activity of 26 kU).The solubilized membrane fraction was dialyzed with 10 mM phosphatebuffer solution (pH 6) for 3 nights, and the obtained insoluble matterwas removed as sediment in a super centrifugation at 10° C. under170,000×g for 90 minutes. The obtained supernatant liquid (solubilizedGDH fraction) contained GDH which showed an activity of 28 kU and aspecific activity of 6.8 U/mg.

(2) Obtainment of Crude Enzyme Solution from Transformants

For purification to be made in Examples and Comparative Exampleshereafter, crude enzyme solution was obtained from each of two kinds oftransformant each derived from a different host.

In the production of the transformants, first, DNA which includessequences for coding manifestation of α, β and γ subunits was obtainedfrom KS1 strain according to a common method.

Next, the obtained DNA was inserted into a vector plasmid, to produce aGDH expression plasmid, which was introduced into host microbes, to maketransformants.

The hosts were provided by Pseudomonas putida KT2440 strain (ATCC 47054)and E. coli bacterium BL21 strain.

When the host was provided by Pseudomonas putida KT2440 strain, RSF1010was used as the vector plasmid for inserting the GDH gene. When the hostwas provided by E. coli bacterium BL21 strain, pTrc99A was used as thevector plasmid. When the E. coli bacterium was used as the host,Cytochrome C maturation (ccm) gene cluster was always inserted. First,DNA which contained sequences for coding E. coli bacterium ccm genecluster was obtained from JM109 strain by using a common method, andinserted into the vector plasmid, to produce ccm expression plasmid. Thevector plasmid employed in this step was pBBR122. Next, transformationwas made by inserting the ccm expression plasmid into the host E. colibacterium which had already engineered with the GDH expression plasmid.

Each transformant was cultured separately from each other.

Pseudomonas putida transformant was cultured under normal aerobicconditions, in 20 L of culture solution. Composition of the culturesolution included 3.2% polypeptone, 2% yeast extract, 0.5% NaCl, 2%glycerol, 0.05 mL/L Adekanol LG-126 (Asahi Denka Co., Ltd., Tokyo) and50 μg/mL streptomycin (pH 7.0). To this culture solution, 200 mL of theprevious culture solution was inoculated and culture was started at 34°C. In four hours since the beginning of culturing, IPTG(Isopropyl-β-D-thiogalactopyranoside) was added at a rate of 0.2 mM, andculturing was continued further for 20 hours, to obtain the targetculture solution. This culture solution was centrifuged by a Sharplesscentrifuge, and approximately 800 g of Pseudomonas putida transformantwas obtained.

E. coli bacterium transformant was also cultured under normal aerobicconditions, in 2 L of culture solution. Composition of the culturesolution included 3.2% polypeptone, 2% yeast extract, 0.5% NaCl, 2%glycerol, 0.05 mL/L Adekanol LG-126 (Asahi Denka Co., Ltd., Tokyo), 50μg/mL ampicillin, and 50 μg/mL kanamycin (pH 7.0). To this culturesolution, 50 mL of the previous culture solution was inoculated andcultured at 30° C. for 29 hours. The culture solution was centrifuged,and approximately 85 g of E. coli bacterium transformant was obtained.

Next, the obtained microbe (transformant) was suspended in 10 mMphosphate buffer solution (pH 8), fragmented in a high pressurehomogenizer (500 bar). Then, Mydol 12 (Kao Corporation, Tokyo) and KClwere added to achieve the final rates of 1% and 0.1 M respectively,followed by steering for 30 minutes. Next, cell solid was separated andremoved by centrifuge (8000×g, 60 minutes at 10° C.), and supernatantliquid was obtained as crude enzyme solution.

In the crude enzyme solutions, Pseudomonas putida transformant was foundto have a GDH activity of 2930 kU, and a specific activity of 22 U/mg.E. coli bacterium transformant was found to have a GDH activity of 259kU, and a specific activity of 10.3 U/mg.

<Measurement of Glucose Dehydrogenation Activity>

Glucose dehydrogenation activity was measured by tracking a redoxreaction of electron acceptor based on glucose dehydrogenation. Theelectron acceptors were provided by 2,6-dichlorophenol-indophenol (DCIP)and phenazine methosulfate (PMS).

Specifically, 900 μL of 47 mM phosphate buffer solution (pH 6.0)containing 20 mM glucose, 2 mM PMS and 0.1 mM DCIP was placed in aspectrophotometer cell, and was pre-incubated at 37° C. for 3 minutes.Next, 0.5-10 μL of the enzyme solution was added. The cell wasimmediately turned upside down to begin reaction, and time coursemonitoring was made for absorption drop at a 600 nm wavelength at 37° C.DCIP has an absorption wavelength of 600 nm and the absorption drop isdue to a redox reaction of the electron acceptors based on glucosedehydrogenation.

Here, calculation of the absorbance was made on the basis that DCIP hada mol absorption coefficient of 4.76 mM/cm. A unit (U) of enzyme wasdefined as an amount by which 1 μM of glucose was oxidized in a minuteunder the standard measurement conditions. The protein concentration wasmeasured by means of UV method, with a protein concentration defined asbeing 1 g/L for absorption of 1 at 280 nm.

Example 1

In this Example, the enzyme solution (solubilized GDH fraction) whichwas obtained from KS1 strain according to the above-described techniquewas purified by using a combination of hydrophobic chromatography andanion exchange chromatography.

The hydrophobic chromatography was performed by using an Octyl sepharose4 Fast Flow column (44 mm ID×20 cm Amersham Bioscience KK) which hadbeen equilibrated with 60 mM phosphate buffer solution (pH6). The columnwas supplied with solubilized GDH fraction (enzyme solution) which wasprepared by adding 1 M phosphate buffer solution (pH 6) to achieve thefinal concentration of 60 mM. Next, 600 mL of 60 mM phosphate buffersolution (pH 6) and 900 mL of 20 mM phosphate buffer solution (pH 8)were passed, and then tightly adsorbed GDH was eluted by supplyingeluent. The eluent was provided by 20 mM phosphate buffer solution (pH8) which contained sodium cholate in dissolved form. The eluent wassupplied at a rate of 15 mL/min so that sodium cholate concentrationwould vary linearly in the range of 0-1 wt %.

As a result, GDH was eluted when the sodium cholate concentration wasapproximately 0.8 wt %, yielding 340 mL of GDH active fraction. Thecollected fraction (Octyl collected fraction) had activity readings of108 U/mg specific activity and 16 kU of total activity.

The anion exchange chromatography was performed by using a Q sepharoseFast Flow column (32 mm ID×12 cm Amersham Bioscience KK) which had beenequilibrated with 20 mM phosphate buffer solution (pH 8). To thiscolumn, the Octyl collected fraction was supplied, and then eluent wassupplied. The eluent was provided by 20 mM phosphate buffer solution (pH8) which contained 1 wt % sodium cholate. The eluent was supplied at arate of 8 mL/min and by a volume of 600 mL.

As a result, GDH was eluted specifically when approximately 300 mL ofthe eluent had been passed, yielding 340 mL of a GDH active fraction.The collected fraction (Q collected fraction) had 770 U/mg specificactivity, and 14 kU total activity.

The following Table 2 summarizes the volume of liquid, total activity,specific activity and yield after each operation. TABLE 2 Liquid TotalSpecific Volume Activity Activity Yield Solubilized GDH 110 mL 28 kU 6.8 U/mg  100% Fraction Octhyl Collected 340 mL 16 kU 108 U/mg 57%Fraction Q Collected 340 mL 14 kU 770 U/mg 50% fraction

Example 2

In this Example, the crude enzyme solution obtained from thetransformant of Pseudomonas putida was purified by using a combinationof hydrophobic chromatography and anion exchange chromatography.

In the hydrophobic chromatography, first, a Phenyl cellulofine column(300 mm ID×10 cm, Chisso Corporation, Tokyo) which had been equilibratedwith 10 mM phosphate buffer solution (pH 8) containing 0.1 M KCl wassupplied with the crude enzyme solution, to let the packing agent holdGDH. Next, 7 L of 10 mM phosphate buffer solution (pH 8) containing 0.1M KCl, and 21 L of 10 mM phosphate buffer solution (pH 8) were passed.Then, tightly adsorbed GDH was eluted by passing eluent. The eluent wasprovided by a 20 mM phosphate buffer solution (pH 8) containing sodiumcholate in dissolved form. The eluent was supplied at a rate of 7 L/minso that sodium cholate concentration would vary linearly in the range of0-1 wt %.

As a result, GDH was eluted when the sodium cholate concentration wasapproximately 0.9 wt %, yielding 7300 mL of an active fraction. Thecollected fraction (Phenyl collected fraction) had activity readings of204 U/mg specific activity and 596 kU total activity.

The anion exchange chromatography was performed by using a Q sepharoseFast Flow column (44 mm ID×20 cm Amersham Bioscience KK) which had beenequilibrated with 10 mM phosphate buffer solution (pH8). The column wassupplied with the Phenyl collected fraction. Note that the phenylcollected fraction was buffer-substituted to 10 mM phosphate buffersolution (pH 8) by using a laboratory module (Asahi Kasei Corporation,Tokyo) which had a 50000 molecular weight cutoff, before being suppliedto the column. Next, the column was supplied with 600 mL of 10 mMphosphate buffer solution (pH 8), and then with eluent. The eluent wasprovided by 10 mM phosphate buffer solution (pH 8) containing 1 wt %sodium cholate. The eluent was supplied at a rate of 10 mL/min.

As a result, GDH was eluted specifically when approximately 1400 mL ofthe eluent had been passed, yielding 313 mL of a GDH active fraction.The collected fraction (Q collected fraction) had 1283 U/mg specificactivity, and 390 kU total activity.

The following Table 3 summarizes the volume of liquid, total activity,specific activity and yield after each operation. TABLE 3 Liquid TotalSpecific Volume Activity Activity Yield Crude Enzyme 1950 mL 2930 kU  22 U/mg 100% Solution Phenyl Collected 7300 mL 596 kU  204 U/mg 20%Fraction Q Collected  315 mL 390 kU 1283 U/mg 13% Fraction

Further, in this Example, the phenyl collected fraction and the Qcollected fraction were subjected to electrophoresis, i.e. SDS-PAGE. TheSDS-PAGE used Tris-Tricine buffer solution and was performed inpolyacrylamide buffer solution in 8-25% gel gradient. Protein whichmigrated in the gel was stained with CBB. Results of the SDS-PAGE areshown in FIG. 1. In this figure, Lane 2 represents CBB-stained Phenylcollected fraction and Lane 3 represents CBB-stained Q collectedfraction.

Example 3

In this Example, the crude enzyme solution obtained from thetransformant of Pseudomonas putida was purified by using a combinationof hydrophobic chromatography and anion exchange chromatography.

The hydrophobic chromatography was performed in the same way as inExample 2. The collected solution was ultracondensed, to yield 70 mL ofphenyl collected fraction, which had a specific activity of 300 U/mg anda total activity of 21 kU.

The anion exchange chromatography was performed by using a QAE-TOYOPEARL550 column (44 mm ID×10 cm, Tohso Corporation, Tokyo) which had beenequilibrated with 10 mM phosphate buffer solution (pH8). The column wassupplied with the phenyl collected fraction, and then with eluent. Theeluent was provided by 10 mM phosphate buffer solution (pH8) containing1 wt % sodium cholate. The eluent was supplied at a rate of 5 mL/min andby a volume of 2000 mL.

As a result, GDH was eluted specifically when approximately 1600 mL ofthe eluent has been passed, yielding 300 mL of active fraction. Thecollected fraction (QAE collected fraction) had 1500 U/mg specificactivity, and 7.8 kU total activity.

The following Table 4 summarizes the volume of liquid, total activity,specific activity and yield after each operation. TABLE 4 Liquid TotalSpecific Volume Activity Activity Yield Crude Enzyme 420 ml 80 kU  59U/mg 100% Solution Phenyl Collected  70 mL 21 kU 300 U/mg 26% FractionQAE Collected 300 mL 7.8 kU  1500 U/mg  10% Fraction

Further, in this Example, the QAE collected fraction was subjected toSDS-PAGE, using the same procedure as used in Example 2, and the proteinwas stained with CBB. Results of the SDS-PAGE are shown in FIG. 1. Inthis figure, Lane 4 represents CBB-stained QAE collected fraction.

Comparative Example 1

In this Comparative Example, the crude enzyme solution obtained from thetransformant of Pseudomonas putida was purified by using a combinationof hydrophobic chromatography and anion exchange chromatography.

The hydrophobic chromatography was performed in the same way as inExample 2, yielding 7200 mL of phenyl collected fraction, which hadspecific activity of 314 U/mg and total activity of 256 kU. In thisComparative Example, however, a portion of the phenyl collected fractionor 1100 mL (Q apply) which represents a total activity of 39 kU wassubjected to the following anion exchange chromatography.

The anion exchange chromatography was performed by using a Q sepharoseFast Flow column (44 mm ID×13 cm Amersham Bioscience KK) which had beenequilibrated with 10 mM phosphate buffer solution (pH 8). The column wassupplied with the Q apply. Thereafter, the column was supplied with 800mL of 10 mM phosphate buffer solution (pH 8) in order to removenon-adsorbent protein. Then, the column was supplied with eluent. Theeluent was provided by 10 mM phosphate buffer solution (pH 8) whichcontained NaCl in dissolved form. The eluent was supplied at a rate of7.5 L/min so that NaCl concentration would vary linearly in the range of0-0.6 M.

As a result, GDH was eluted at two NaCl concentration levels of 0.25 Mapprox. and 0.4 M approx., yielding 140 mL and 360 mL of activefractions respectively. Each of the active fractions (Q collectedfraction (1) and Q collected fraction (2)) were subjected to activitymeasurement. The Q collected fraction (1) had specific activity of 600U/mg and total activity of 4.5 kU. The Q collected fraction (2) hadspecific activity of 432 U/mg and total activity of 12 kU.

The following Table 5 summarizes the volume of liquid, total activity,specific activity and yield after each operation. TABLE 5 Liquid TotalSpecific Volume Activity Activity Yield Crude Enzyme 1250 mL 1066 kU  36 U/mg 100% Solution Phenyl Collected 7200 mL 256 kU  314 U/mg 24%Fraction Q Apply 1100 mL 39 kU 314 U/mg 24% Q Collected Fraction (1) 140 mL 4.5 kU  600 U/mg 3% Q Collected Fraction (2)  360 mL 12 kU 432U/mg 7%

Further, in this Comparative Example, the phenyl collected fraction andthe Q collected fraction (2) were subjected to SDS-PAGE using the sameprocedure as used in Example 2, and the protein was stained with CBB.Results of the SDS-PAGE are shown in FIG. 2. In this figure, Lane 2represents the phenyl collected fraction and Lane 3 represents the Qcollected fraction (1).

Comparative Example 2

In this Comparative Example, a portion of the phenyl collected fractionobtained in Comparative Example, or the 2100 mL (QAE apply) whichrepresents the total activity of 74 kU, was subjected to anion exchangechromatography, to obtain purified GDH.

The anion exchange chromatography was performed by using a QAE-TOYOPEARL550 column (44 mm ID×10 cm Tohso Corporation, Tokyo) which had beenequilibrated with 10 mM phosphate buffer solution (pH 8). The column wasfirst supplied with the QAE collected fraction (74 kU total activity).Next, 800 mL of 10 mM phosphate buffer solution (pH 8) was supplied toremove non-adsorbent protein. Then, the column was supplied with eluent.The eluent was provided by 10 mM phosphate buffer solution (pH 8)containing dissolved KCl. The eluent was supplied at a rate of 5 mL/minso that KCl concentration would vary linearly in the range of 0-1M.

As a result, GDH was eluted at two KCl concentration levels of 0.23 Mapprox. and 0.43 M approx., yielding 200 mL and 400 mL of activefractions respectively. Each of the active fractions (QAE collectedfraction (1) and QAE collected fraction (2)) were subjected to activitymeasurement. The QAE collected fraction (1) had specific activity of 399U/mg and total activity of 7.5 kU. The QAE collected fraction (2) hadspecific activity of 217 U/mg and total activity of 6.4 kU.

The following Table 6 summarizes the volume of liquid, total activity,specific activity and yield after each operation. TABLE 6 Liquid TotalSpecific Volume Activity Activity Yield Crude Enzyme Solution 1250 mL1066 kU   36 U/mg 100% Phenyl Collected Fraction 7200 mL 256 kU  314U/mg 24% QAE Apply 2100 mL 74 kU 314 U/mg 24% QAE Collected Fraction (1) 200 mL 7.4 kU  399 U/mg 2% QAE Collected Fraction (2)  400 mL 6.4 kU 217 U/mg 2%

Further, in this Comparative Example, the QAE collected fraction wassubjected to SDS-PAGE using the same procedure as used in Example 2, andthe protein was stained with CBB. Results of the SDS-PAGE are shown inFIG. 2. In this figure, Lane 4 represents the QAE collected fraction.

Example 4

In this Example, the crude enzyme solution obtained from thetransformant of E. coli bacterium was purified by using a combination ofhydrophobic chromatography and anion exchange chromatography.

The hydrophobic chromatography was performed under the same conditionsas in Example 1, using an Octyl sepharose 4 Fast Flow column.

As a result, GDH was eluted at the sodium cholate concentration ofapproximately 0.8 wt %, yielding 220 mL of GDH active fraction. Thecollected fraction (Octyl collected fraction) had specific activity of503 U/mg and total activity of 149 kU.

The anion exchange chromatography was performed by using a Q sepharoseFast Flow column and under the same conditions as in Example 1.

As a result, GDH was eluted specifically when approximately 500 mL ofthe eluent had been passed, yielding 175 mL of GDH active fraction. Thecollected fraction (Q collected fraction) had 1147 U/mg specificactivity, and 50 kU total activity.

The following Table 7 summarizes the volume of liquid, total activity,specific activity and yield after each operation. TABLE 7 Liquid TotalSpecific Volume Activity Activity Yield Crude Enzyme Solution 520 mL 259kU 10.3 U/mg  100% Octhyl Collected Fraction 220 mL 149 kU 503 U/mg 58%Q Collected Fraction 175 mL  50 kU 1147 U/mg  19%

Further, in this Example, the Q collected fraction was subjected toSDS-PAGE using the same procedure as used in Example 2, and the proteinwas stained with CBB. Results of the SDS-PAGE are shown in FIG. 3. Inthis figure, Lane 3 represents the Q collected fraction according to thepresent Example. In FIG. 3, the Q collected fraction (Lane 2) accordingto Example 1 and the Q collected fraction (Lane 4) according to Example2 are also subjected to the electrophoresis.

<Discussion on the Results>

As understood from Table 2 through Table 7, GDH has a higher finalspecific activity and is purified more efficiently when the eluent isprovided by cholate (Example 1 through 4) than when the eluent isprovided by NaCl or KCl and GDH is purified through gradient elution(Comparative Example 1 and 2). This is also indicated in FIG. 1 and FIG.2. Specifically, GDH derived from the KS1 strain includes α, β and γsubunits, and their molecular weights in the SDS gel-electrophoreticmigration under reducing environment were approximately 60 kDa, 43 kDaand 14 kDa respectively. With this point in mind, FIG. 1 and FIG. 2 willreveal that the collected fractions in Example 2 and Example 3 havewider bands which represent α, β and γ subunits, than the correspondingbands found in the collected fractions in Comparative Examples 1 and 2,while having smaller portions of proteins which have different molecularweights. Therefore, when purifying GDH by using a combination ofhydrophobic chromatography and anion exchange chromatography, use ofsodium cholate for eluting GDH will lead to efficient purification ofGDH.

Further, as understood from FIG. 3, crude enzyme derived from atransformant of host E. coli bacterium shows a narrower band for the γsubunit but wider bands for α and β subunits than other crude enzymesolutions. E. coli bacterium is advantageous in that it is inexpensive,easily available and has superb self-duplicating capability. Therefore,in terms of industrial application, the method of obtaining a crudeenzyme from a transformant hosted by E. coli bacterium and purifyingthis crude enzyme to obtain GDH will be useful.

1. A method for purifying a target protein from a protein solutioncontaining the target protein by using liquid chromatography, whereinthe liquid chromatography comprises: a first step of introducing theprotein solution into a column filled with a packing agent and causingthe packing agent to hold the target protein; and a second step ofeluting the target protein by using an eluent containing ahydroxy-cholate.
 2. The method for purifying protein according to claim1, wherein the target protein contains an electron transfer protein. 3.The method for purifying protein according to claim 2, wherein thetarget protein is provided by a glucose dehydrogenase containing aprotein having glucose dehydrogenation activity.
 4. The method forpurifying protein according to claim 1, wherein the packing agent isprovided by an ion-exchange gel.
 5. The method for purifying proteinaccording to claim 4, wherein the ion-exchange gel contains a quaternaryammonium group as an ion-exchange group.
 6. The method for purifyingprotein according to claim 3, wherein the electron transfer protein hasa molecule weight of approximately 43 kDa in SDS-gel electrophoresisunder a reducing environment, the protein which has glucosedehydrogenation activity having a molecule weight of approximately 60kDa in SDS-gel electrophoresis under a reducing environment.
 7. Themethod for purifying protein according to claim 1, wherein thehydroxy-cholate comprises a cholate.
 8. The method for purifying proteinaccording to claim 1, wherein the hydroxy-cholate in the eluent ismaintained at a constant concentration in the elution of the targetprotein from the packing agent.
 9. The method for purifying proteinaccording to claim 8, wherein the concentration of the hydroxy-cholatein the eluent is selected from a range of 0.5 through 2.5 wt %.
 10. Themethod for purifying protein according to claim 3, wherein the glucosedehydrogenase is produced by a microorganism belonging to the genusBurkholderia.
 11. The method for purifying protein according to claim10, wherein the microorganism belonging to the genus Burkholderia isprovided by Burkholderia cepacia KS1 strain (FERM BP-7306).
 12. Themethod for purifying protein according to claim 3, wherein the glucosedehydrogenase is produced by a transformant, the transformant beingproduced by engineering a host microorganism with a DNA from amicroorganism belonging to the genus Burkholderia for coding theelectron transfer protein and the protein active against glucose. 13.The method for purifying protein according to claim 12, wherein the hostmicroorganism is provided by Pseudomonas putida.
 14. The method forpurifying protein according to claim 12, wherein the host microorganismis provided by E. coli bacterium.
 15. A method for purifying glucosedehydrogenase using a combination of hydrophobic chromatography andanion exchange chromatography, wherein the hydrophobic chromatographyincludes: a step of causing a stationary phase to hold the glucosedehydrogenase; a step of eluting unnecessary proteins; and a step ofeluting the glucose dehydrogenase by using an eluent containing ahydroxy-cholate, the anion exchange chromatography including: a step ofcausing a stationary phase to hold the glucose dehydrogenase; and a stepof eluting the glucose dehydrogenase by using an eluent containing ahydroxy-cholate.
 16. The method for purifying glucose dehydrogenaseaccording to claim 15, wherein concentration of the hydroxy-cholate inthe eluent is varied with time in the hydrophobic chromatography,concentration of the hydroxy-cholate in the eluent being kept at aconstant level in the elution of the glucose dehydrogenase in the anionexchange chromatography.
 17. The method for purifying glucosedehydrogenase according to claim 16, wherein the anion exchangechromatography is carried out after the hydrophobic chromatography. 18.The method for purifying glucose dehydrogenase according to claim 15,wherein the glucose dehydrogenase is produced by a microorganismbelonging to the genus Burkholderia.
 19. The method for purifyingglucose dehydrogenase according to claim 18, wherein the microorganismbelonging to the genus Burkholderia is provided by Burkholderia cepaciaKS1 strain (FERM BP-7306).
 20. The method for purifying glucosedehydrogenase according to claim 15, wherein the glucose dehydrogenaseis produced by a transformant, the transformant being produced byengineering a host microorganism with a DNA from a microorganismbelonging to the genus Burkholderia for coding the glucosedehydrogenase.
 21. The method for purifying glucose dehydrogenaseaccording to claim 20, wherein the host microbe is provided byPseudomonas putida.
 22. The method for purifying glucose dehydrogenaseaccording to claim 20, wherein the host microbe is provided by E. colibacterium.
 23. The method for purifying glucose dehydrogenase accordingto claim 15, wherein the anion exchange chromatography uses anion-exchange gel containing a quaternary ammonium group as anion-exchange group, the hydroxy-cholate being provided by a cholate.